The liver and kidney excrete from the body a wide array of positively charged organic molecules of physiological, pharmacological and toxicological significance. Carrier-mediated secretion of these "organic cations" (OCs), particularly by the kidney, has a profound influence on the pharmacokinetics of these compounds and, importantly, OC secretion is the site of many clinically significant drug-drug interactions. Although the molecular basis for the first (i.e., entry) step in renal and hepatic OC secretion is well defined, the active and rate-limiting step (i.e., substrate exit from cells into the tubular filtrate or bile) is poorly understood. The physiological hallmark of the exit step in OC secretion, as determined in studies with membrane vesicles and intact renal tubules, is carrier-mediated OC/H+ exchange, and a novel group of transporters (the Multidrug And Toxin Extruders, the MATEs) was recently cloned from human kidney and liver (MATE1 and MATE-2K) that displays this "physiological fingerprint." Strong evidence supports the growing consensus that MATEs are major contributors to renal and hepatic OC secretion. Despite the large number of MATE transporters that have been identified (only two in humans, but 750+ in prokaryotes, fungi, and plants), and the likelihood that MATE transporters represent the primary driving element in active renal and hepatic secretion of cationic drugs, virtually nothing is known about the molecular characteristics of these proteins. In this proposal, we describe hypothesis-driven experiments that will identify the substrate-binding region of MATE transporters and determine the secondary structure and helical organization of these proteins. The studies use a combination of site-directed mutagenesis (cysteine scanning and chimeras), and proteomic methods (photoaffinity labeling and mass spectrometry), to gain structural insight into the molecular basis of MATE transport activity. These studies will be essential for establishing models that accurately predict and, ideally, preempt unwanted interactions of cationic drugs in both the kidney and liver.